The high speed supernatant of Novikoff hepatoma cells contains factors which are capable of increasing the rate of DNA synthesis by the homogeneous beta-polymerase. These factors have been purified to varying degrees: Factor I has not been purified yet; Factor II is 70 percent homogeneous; Factor III is pure; and Factor IV is 50 percent purified. The proposed research is aimed at: 1) identifying any and all factors participating in or regulating DNA synthesis; 2) purifying each factor to homogeneity; 3) characterizing each factor with regard to enzymatic activity, molecular weight and subunit structure, stoichiometry of its action on in vitro DNA synthesis, binding to DNA or DNA polymerase, interaction with other factors, subcellular localization and amounts present under different physiological conditions; 4) clearly establishing a role for each in DNA synthesis in vivo. The beta-polymerase as extracted from Novikoff cells exists as an aggregate of proteins sedimenting at 6-8S (7.3S average). The homogeneous enzyme sediments at 3.3S and intermediate forms of 4.1S and 5.8S can be experimentally generated. Factor IV has been shown to combine with the 3.3S enzyme to form the 4.1S intermediate. The other two proteins needed to form the 5.8S and 7.3S forms are being sought using change in sedimentation in sucrose gradients or change in molecular weight in Sephadex gels as the assay. Preparation of antibody to the polymerase and each factor will be attempted. Such antibody will be used to determine the time of synthesis in the cell cycle, amount present in normal vs tumor cells, and subcellular localization using fluorescent and radiolabelled antibody. BIBLIOGRAPHIC REFERENCES: G.S. Probst, D.M. Stalker, D.W. Mosbaugh and R.R. Meyer (1975), Stimulation of DNA polymerase by factors isolated from Novikoff hepatoma, Proc. Nat. Acad. Sci., 72, 1171. R.R. Meyer, D.M. Stalker, D.W. Mosbaugh, G.S. Probst and B.F. Kluve (1975), Identification, purification and properties of accessory proteins for DNA polymerase isolated from the Novikoff hepatoma, Proc. Int. Conf. Eukaryotic DNA Polymerases, May, 1975 (abstract).